PRAC 2 - Only 2 first question PDF

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Only 2 first question...

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1. Did you manage to isolate DNA from E. coli? Describe below what it looks like.

Yes. We managed to isolate DNA from E.coli. It includes 2 layers from the centrifugation. One solid DNA layers and one transparent liquid layer which look like water. The solid form has muddy brown color.

2. What would be the next steps if you were going to use this DNA sample for further analysis?

Think what factors you would need to know to move forwards.

We have to check the quality and quantity of DNA sample. Particularly, checking whether that sample is pure or not, how the contamination of remaining DNA. We also need to base on the value of wavelength to check the purity of DNA sample. For example, 280 nm is used for checking RNA or Protein contaminants

Investigation 2: Quantification of your DNA sample

This practical is a LabArchives intensive practical. When we mark your LabArchives notebook after week 13, we will definitely select an item from one of the Lab Archives intensive practicals (prac 2, 4, 11).

Although we know that your LabArchives is not formally marked until week 13, your LabArchives for practical 2 has not been attempted, or is very incomplete. As there is no paper version of the practical it is important that you are completing this practical on LabArchives DURING the practical session. If you are having trouble with how to enter data/text/images into LabArchives, please talk to one of your demonstrators or contact [email protected]

Please ignore this comment if you have a special consideration approved for this practical. When we mark your LabArchives after week 13, we will not mark any practicals for which you have a special considerations approved. Please note however, all practical content is still assessable in the post module quizzes and final exams Complete the table below with the protocol steps that correspond to each sentence of the legend.

Legend sentence starting with: Protocol Step Numbers

DNA was isolated from E. coli culture (~0.1 g wet weight/mL, suspended in 0.1 M NaCl, 10 mM EDTA, pH 8) using a commerical DNA extraction kit (BioRad Aurum) according to the manufacturer's instructions. None Cells were collected by centrifugation (12000 x g) for 2 mins and lysed in Nuclei Lysis Buffer containing 0.5% (w/v) SDS at 80°C for 5 min. 1,2,3 RNase (100 μg/mL) was added with further incubation at 37°C for 30 min. The solution was then vortexed with Protein Precipitation Solution.

4,5

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After incubation at 0oC for 5 min, the lysate was centrifuged (12000 x g) for 3 min and the supernatant was added to isopropanol at a ~1:2 ratio. 7,8,9 The precipitated DNA was collected by centrifugation (12000 x g) for 2 min, washed briefly in 70% (v/v) ethanol and then dissolved in Rehydration solution (10 mM Tris, 1 mM EDTA, pH 7.4) 10,11,12,13,14,15,16 The yield and purity was assessed by UV spectrophotometry.

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